1. BIOMARKERS Diagnostics and Prognostics

2. OMICS Molecular Diagnostics: Promises and Possibilities, p. 12 and 26

4. Biomarkers are defined as endogenous or injected molecules whose presence or metabolism correlates with important disease related physiological processes and disease outcomes (Ferber, 2002). They should be identified or defined molecular entities to facilitate comparison across laboratories and technology platforms. A biomarker is a characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention.

5. According to the US National Institutes of Health’s (NIH) Working Group and the Biomarkers Consortium, a biomarker is a characteristic that is objectively measured as an indicator of normal biological processes, pathogenic processes, or a pharmacological response to a therapeutic intervention (http://www.biomarkersconsortium.org).

6. TOOLS FOR BIOMARKER DISCOVERY Genomics – Ultra-fast DNA sequencing – whole human genome or large number of samples – Connect to genome-wide associated studies (GWAS) for the discovery of disease-specific mutations

7. TOOLS FOR BIOMARKER DISCOVERY Transcriptomics – Changes in gene expression levels in tissue and cells, i.e., microarrays – Using as many as 10,000 probes can compare numerous patient/normal populations, disease states and sample types

8. TOOLS FOR BIOMARKER DISCOVERY Transcriptomics – identify genes differentially expressed Oncotype DX breast cancer multigene (21 genes) expression evaluated in 14 clinical studies involving > 4,000 breast cancer patients worldwide – RT PCR of RNA from tumor tissue; 21 genes Oncotype DX for colon cancer – 12 genes Both used to predict recurrence

9. Oncotype DX Assay RNA extracted from piece of formalin fixed, paraffin embedded tissue, i.e., part of tissue used for pathology Treated with Dnase 1 Measure total RNA concentration Reverse transcription followed by quantitative TaqMan ® (Roche Molecular Systems, Inc.) Each of 16 genes is measured in triplicate and then normalized relative to a set of five reference genes. Real Time PCR to quantitate 21 genes in the sample

10. STEPS USED TO DEVELOP ASSAY optimize RT-PCR technology – for high-throughput, real-time quantitation of specific RNA in FPET – to be reproducible regardless of the variability inherent in tumor block 25,000 human genes - identify 250 candidate genes possibly associated with breast cancer tumor behavior

12. Narrowing 250 candidate genes – Analyzed tissue from 447 patients ((3 independent clinical studies) – Identified panel of genes strongly correlated with distant recurrence-free survival – Selected 16 cancer genes and 5 reference genes to normalize amounts of cancer genes – Developed Recurrence Score result calculation to combine individual gene results into a single result

13. BIOLOGICAL BASIS

14. RECURRENCE SCORE = number between 0 and 100 that corresponds to a specific likelihood of breast cancer recurrence within 10 years of the initial diagnosis < 186.8% risk of recurrence 18-3014.3% > 3030.5% Guides treatment plan: radiation, standard chemotherapy, monoclonal antibody therapy

16. ONCOTYPE DX Validated only for patients with – node-negative, estrogen-receptor-positive invasive breast cancer (spread beyond the ducts)

17. Validated Oncotype DX assay gene panel and Recurrence Score result calculation: – large, independent, multicenter clinical trial – large population-based case-control study – Demonstrated consistent statistical link to distant breast cancer recurrence, as well as robust predictive power regarding chemotherapy benefit www.oncotypedx.com

18. NARROWING AND INTERPRETATION REQUIRE Robust bioinformatics and statistical capabilities Appropriate data management, ability to extract knowledge from massive amounts of data, and availability of functional information for data interpretation Data – Management – Analysis – Interpretation

19. GENOMICS APPLICATIONS improve understanding of basic biological processes and their diversity delineate mechanisms of efficacy and toxicity of xenobiotic compounds (e.g., drug, dietary supplements, and environmental agents) understand disease processes generate predictive models or molecular classifiers to provide better predictive and diagnostic accuracy

20. GENOMICS APPLICATIONS animal husbandry, species, aging, and gender differences, e.g., genes expressed in liver distinguish genders, distinguish different albino rats Drug metabolism, drug mechanism of action, pharmacotoxicity, Differences/similarities of in vitro/in vivo cells, identify stem cell characteristics Kidney disease, heart failure

21. SEQUENCING – HOW FAST 10 years to develop draft of 1 st human genome Now it takes 10 days

22. FLOW OF INFORMATION

23. PROTEOMICS Biomarkers, proteome and protein-protein interaction data, pathways and 3-D structures availability of gene and genome sequence databases and the discovery and development of protein ionization methods Identify expression of proteins as a function of cell or tissue state Widely applied Mass spectrometry (MS) commonly used – designed to identify peptides not proteins Identify thousands of proteins within complex samples (such as blood, urine, tissue, etc.) identify and quantitate differences in proteins between comparative samples (e.g., healthy versus diseased) Within hours, e.g., approximately 1000 peptides can be confidently identified in a single one-hour LC-MS2 experiment

24. MASS SPECTROSCOPY Converts compounds into ions – move about and manipulate by external electric and magnetic fields Parts: – Ion source: converts into (usually) cations by loss of electron – Mass analyzer: sorts and separates ions according to mass and size; operates in a near vacuum

26. Prior to MS other techniques used for preliminary separation – 2D gel electrophoresis – Liquid chromatography – ….

28. END